Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Abstract

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Is Circulating Tumor DNA (Ctdna) Use Ready For Prime Time? Applications and Challenges of Ctdna in the Era of Precision Oncology

“…NGS is highthroughput sequencing that involves processing millions of DNA fragments. The ctDNA platform in our study utilized massively parallel and deep digital sequencing from a blood sample [7]. In contrast, tissue NGS involved parallel DNA sequencing from formalin-fixed or paraffin-embedded specimens.…”

Review of Clinical Next-Generation Sequencing

“…20,26,27 This testing is often referred to as a liquid biopsy. The potential applications of sequencing ctDNA include screening or diagnosis of cancer, monitoring for progression or relapse, and guiding therapy for a patient with a known cancer diagnosis.…”

“…Most research studies have evaluated the ability of ctDNA sequencing to detect somatic mutations in patients with known cancer and the ability to monitor disease. [26][27][28][29] Monitoring a known mutation by sequencing of ctDNA has been shown in several studies to correlate with relapse/progression of disease. 29,30 Also, using detection of mutations in ctDNA to help guide therapy for a patient with a known tumor has shown utility, for example, tyrosine kinase inhibitor response with epidermal growth factor receptor (EGFR)-activating mutations in lung cancer.…”

“…29,30 Also, using detection of mutations in ctDNA to help guide therapy for a patient with a known tumor has shown utility, for example, tyrosine kinase inhibitor response with epidermal growth factor receptor (EGFR)-activating mutations in lung cancer. 26,27,31,32 Although ctDNA may have lower sensitivity for detecting mutations than testing tumor tissue, the most common clinical applications of ctDNA appear to be for patients with metastatic cancers when there is insufficient tissue for testing and a repeated biopsy would be associated with significant morbidity and mortality, and when testing of ctDNA is a reasonable alternative. 26,27,31 Using ctDNA to screen for or diagnose early-stage cancers is more problematic.…”

“…26,27,31,32 Although ctDNA may have lower sensitivity for detecting mutations than testing tumor tissue, the most common clinical applications of ctDNA appear to be for patients with metastatic cancers when there is insufficient tissue for testing and a repeated biopsy would be associated with significant morbidity and mortality, and when testing of ctDNA is a reasonable alternative. 26,27,31 Using ctDNA to screen for or diagnose early-stage cancers is more problematic. 33 Most studies of patients with known cancer have not contained normal controls, but a limited number of targeted sequencing studies have shown some degree of mutation detection (false positives), albeit usually at a low level, in normal controls.…”